冷冻样品制备.pdf

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1、PreparingCryogenicSpecimenswithFEIVitrobotElectronMicroscopyCore,UniversityofUtahCriticalstepsmarkedwithasterisk1.TurnonVitrobotand(OPTIONAL)settodesiredtemperatureandhumidity.-turnoncompressedair,airpressureshouldbeabout90psi-ifyouwanttohavehigherthanambienthumidity,youwillp

2、robablyneedtoaddwatertothehumidifier2.Coolcryogeniccontainer-pourliquidnitrogenintobrasscupandletoverflowuntilremainderofcontainer(liquidnitrogenreservoir)isfull3.Makeliquidethane(orpropane)*REMOVEANYSOURCETHATMIGHTINITIATECOMBUSTION-afterliquidnitrogenhasdisappearedfromthebr

3、asscup,thecontainershouldbecoldenoughtomakeliquidethane-inserttipofhose(glassPasteurpipetworkswellasatip)intobrasscontainer-turnongas-movingtipmaypreventapartofthebrasscupfrombecomingtoowarm-besurethatthebrasscupisthermallyconnectedtotheliquidnitrogenreservoir4.Putcleanpadsof

4、filterpaperonblotters(inenvironmentalchamber)-usecleangloves5.Duringsteps6–10ensurethata.thereservoircontainsareasonableamountofliquidnitrogen.b.theethane(orpropane)remainsliquidoraslush.Ificeformswarmbyinvertingthethermalconnector(thatconnectsbrasscupandliquidnitrogenreservo

5、ir.6.PlacegridontweezersandinserttweezersontoVitrobotshaft-Click"PlaceGrid"7.Setappropriatesettingsforspecimen-setblottingtime-setoffset(whereblottingpadwillcontactspecimen)-(OPTIONAL)setwaittime-Notethatthesesettingsshouldbedonebeforecontinuing.Otherwisetherunwillhavetobeabo

6、rted.8.ContinueaspromptedbyVitrobotsoftware-applysampletogridaftergridisdroppedwithintheenvironmentalchamber(afterpressing"StartProcess"button)9.Afterspecimenisfrozen,removetweezersfromshaftandplacespecimeningridbox*specimenmustbekeptcold(preferablysubmergedinliquefiedethaneo

7、rnitrogen10.Transfergridboxtocryo-transferstationorliquid-nitrogenstoragedewar11.TurnoffVitrobot—includingcompressedair112.Tips-Blottingisnotnecessarilyreproducibleevenforestablishedsamples:-usemultipleblottingtimesandgridtypes,especiallyforanewsampleorfornewconditions,suchas

8、changesinbuffer,saltconcentration,ortemperature-Ifyouhavevitrifiedth